Glucagon-like peptide-1 receptor (GLP-1R), a member of family B G protein-coupled receptors (GPCRs), is a highly attractive therapeutic target for type 2 diabetes. It has been shown clinically that the activation of GLP- 1R has many anti-diabetic effects, including potentiating insulin biosynthesis, increasing glucose-dependent insulin secretion, inhibiting pancreatic -cell apoptosis and promoting -cell neogenesis. A few peptide agonists of GLP-1R have been brought to market in the past decade, but their clinical utility is limited by their short half-life, side effects and lack of oral bioavailability. A long asting, orally active small molecule drug for GLP-1R would significantly advance the state of the art in diabetes treatment. However, the development of such small molecule drugs has long been hindered by a lack of structural information about GLP-1R. The ultimate goal of this proposal is to obtain a high-resolution crystal structure of active-state GLP-1R in order to facilitate structue-based small molecule drug discovery. In Phase I, we will develop methods and tools to produce milligram quantities of high quality, active-state GLP-1R for crystallography studies that will be performed in Phase II. A major challenge in GPCR crystallization is to obtain large quantities of pure, stable receptor protein in its active-state, which in the case of GLP-1R is known to be unstable without additional stabilizing molecules. This proposal shows strong preliminary evidence that a novel method developed at ConfometRx enables us to form a stable GLP-1R-Gs protein complex in milligram quantities and at sufficient purity for crystallization. We will alo generate conformationally selective nanobodies that may further stabilize the GLP-1R-Gs complex or act as G protein surrogates to stabilize the active-state GLP-1R for crystallography. The major focus of Phase II is the crystallization and structure determination of active-state GLP-1R. We will pursue a comprehensive strategy to increase the chances of obtaining diffraction-quality crystals, such as crystallization in different lipid environments and crystal lattice contact enhancement by T4 lysozyme and nanobody techonologies. Once we have obtained the crystal structure of GLP1-R, it will allow us to perform in silico screening of small-molecule agonists or positive allosteric modulators. Novel lead compounds will be experimentally validated and subjected to structure-based optimization. Commercialization of this research will be achieved by providing data and services to pharmaceutical companies working on GLP-1R drugs. This proposal is in response to the NIDDK's RFA-DK-14-008, Reagents for Glucagon and Incretin Research.